Control of septin filament flexibility and bundling by subunit composition and nucleotide interactions

Abstract

Septins self-assemble into heteromeric rods and filaments to act as scaffolds and modulate membrane properties. How cells tune the biophysical properties of septin filaments to control filament flexibility and length, and in turn the size, shape, and position of higher-order septin structures, is not well understood. We examined how rod composition and nucleotide availability influence physical properties of septins such as annealing, fragmentation, bundling, and bending. We found that septin complexes have symmetric termini, even when both Shs1 and Cdc11 are coexpressed. The relative proportion of Cdc11/Shs1 septin complexes controls the biophysical properties of filaments and influences the rate of annealing, fragmentation, and filament flexibility. Additionally, the presence and apparent exchange of guanine nucleotide also alters filament length and bundling. An Shs1 mutant that is predicted to alter nucleotide hydrolysis has altered filament length and dynamics in cells and impacts cell morphogenesis. These data show that modulating filament properties through rod composition and nucleotide binding can control formation of septin assemblies that have distinct physical properties and functions.

Interplay of septin amphipathic helices in sensing membrane-curvature and filament bundling

“…Shs1-capped octamers displayed a similar membrane curvature preference to that of Cdc11-capped octamers with the highest adsorbance on beads 1 µm in diameter (κ = 2 μm -1 ) ( Figure 2B) (Cannon et al, 2019). This is consistent with the behavior of tandem AH domains (Figure 2A and Cannon et al, 2019) and our previous observation where Cdc11-and Shs1-capped octamers were mixed and preferentially adsorbed onto membrane curvatures 1 µm in diameter (Khan et al, 2018). It was unclear why addition of two more AH domains did not alter membrane-curvature sensitivity.…”

“…Why does Shs1 harbor a curvature-sensing AH domain? Within the septin complex Cdc12 resides at the penultimate position, with the terminal subunit being either Cdc11 or Shs1 (Bertin et al, 2008;Versele et al, 2004) and there is no evidence of "chimeric" octamers with Cdc11/Shs1 on either end (Garcia et al, 2011;Khan et al, 2018;Weems and McMurray, 2017). Thus, Cdc11-capped complexes possess two AH domains, spaced apart by ~24 nm (the distance between the Cdc12 subunits) while Shs1-capped complexes have four AH domains, altering the valency and spacing of AHs within assemblies.…”

Membrane binding controls ordered self-assembly of animal septins

“…Septins are a conserved family of cytoskeletal proteins [1] capable of forming filamentous scaffolds at the cell cortex that participate in many processes such as cytokinesis, cell-cell adhesion, and phagocytosis [2][3][4][5]. Most of what we currently know about the molecular mechanisms by which septins function comes from studies of the budding yeast cell S. cerevisae, where septins form hetero-octamers [6][7][8][9][10] that form paired filaments [11][12][13]. During budding, septins form a collar encircling the bud neck that acts as a scaffold to recruit proteins necessary for cell division [14][15][16], and that restricts lateral diffusion of transmembrane proteins [17][18][19].…”

An amphipathic helix enables septins to sense micron-scale membrane curvature

“…Septins carry out these functions by assembling into heteromeric, rod-shaped, non-polar complexes which can anneal end-on to polymerize into filaments at the plasma membrane (Field et al, 1996;John et al, 2007;Sirajuddin et al, 2007;Bertin et al, 2008). Budding yeast possess five mitotic septins that assemble into hetero-octamers in which the terminal subunit is either Cdc11 or Shs1 (Garcia et al, 2011;Khan et al, 2018). Purified recombinant septins from yeast and humans preferentially adsorb onto micrometer curvatures in the absence of any cellular factors (Bridges et al, 2016) indicating that curvature sensing is a conserved feature of the septin cytoskeleton.…”